Phototropin localization and interactions regulates photophysiological processes inChlamydomonas reinhardtii

Abstract Phototropin, a blue-light sensing serine/threonine kinase, plays a pivotal role in regulating diverse photophysiological processes in both plants and algae. InChlamydomonas reinhardtii, phototropin (CrPhot) localizes to the eyespot and flagella, coordinating key cellular functions such as phototaxis, photosynthesis, gametogenesis, and chlorophyll biosynthesis. While previous research has identified phototropin interactions with signaling proteins such as channelrhodopsins and light-harvesting complex proteins, many aspects of its interaction network and regulatory mechanisms remain unresolved. In this study, we explored novel interacting protein partners of phototropin and their roles in modulating its regulatory functions inChlamydomonas reinhardtii. Employing a suite of intraflagellar transport (IFT) mutants ofC. reinhardtiisuch as IFT172, IFT52, IFT88, IFT139, kinesin/dynein, CEP290 etc., we elucidate that phototropin localization within the flagella and eyespot is IFT-mediated. Our study highlights interaction of phototropin with other photoreceptors-channelrhodopsins (ChR1 and ChR2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and signaling protein-14-3-3. CRISPR-Cas9 knockouts of phototropin showed reduced ChR1, 14- 3-3 levels and exhibited impaired photomotility. Moreover, two LOV-domain containing histidine kinases, LOV-HK1 and LOV-HK2, were identified inC. reinhardtii. Gene expression of LOV-HK1 and LOV-HK2 were found to be elevated in UV-light inC. reinhardtiiand their genes expression was found to be altered in phototropin CRISPR-Cas9 knockouts. This study provides new insights into phototropin signalosome and highlights molecular mechanisms governing its function. The research outcomes advances our understanding of phototropin trafficking and signal modulation inChlamydomonas reinhardtii, and sets the stage for further exploration into the broader physiological roles of phototropin in cellular responses. Graphical abstract Phototropin, a blue-light receptor inChlamydomonas reinhardtii, localizes to the flagella and eyespot, mediates phototaxis and photosynthesis. Its trafficking is mediated by intraflagellar transport (IFT) machinery, with mutations in IFT components (kinesin, dynein, IFT172, IFT52, IFT88, IFT139, CEP290) disrupting phototropin localization. Phototropin interacts with other photoreceptors like channelrhodospins (ChR1/2), chlamyopsin 6, LOV-histidine kinases (LOV-HK1, LOV-HK2) and signaling proteins (14-3-3), coordinating light-driven responses. These findings underscore the details of phototropin trafficking and phototropin signaling impacting light-induced physiological processes inC. reinhardtii. Highlights • Phototropin localizes in eyepot and flagella inChlamydomonas reinhardtii. • Intraflagellar transport (IFT) mutants ofC. reinhardtiisuggest role of different IFT proteins in phototropin trafficking and localization. • Phototropin interacts with other photoreceptors (ChR1 & ChR2, COP6, LOV-HK1 & LOV-HK2) and signaling proteins (14-3-3), contributing to various physiological processes. • CRISPR-Cas9 knockouts of phototropin showed reduced 14-3-3 protein content and photomotility response inC. reinhardtii.